Project Summary Over 200 million people worldwide are chronically exposed to arsenic in drinking water at concentrations above the EPA or World Health Organization safety standard. There is strong experimental and epidemiological evidence that low levels of arsenic in combination with other environmental insults such as ultraviolet radiation (UVR) increases carcinogenesis, suggesting arsenic is a co-carcinogen in humans. However, little is known about the molecular mechanisms of arsenic co-carcinogenesis or effective strategies for prevention of arsenic- augmented cancers. Recent advances in analysis of next generation sequencing have given rise to powerful tools to define distinct mutational signatures in tumors that identify specific defects in DNA repair processes or carcinogenic exposures as part of cancer etiology. In current proposed study we will apply this new technology to advance our understanding of arsenic as a co-carcinogen when combined with the DNA damaging UVR. We have published an extensive body of work demonstrating that arsenic interferes with the zinc finger motifs of select DNA repair proteins leading to decreased repair capacity and increased DNA damage and mutations that are alleviated by zinc. A preliminary mutation pattern analysis of normal human keratinocytes exposed to 0.1 M arsenite, UVR, or both revealed that this low concentration of arsenite was sufficient to enhance UVR- induced C>T mutations and zinc supplement reduced C>T mutations suggesting a potential intervention. Furthermore, the mutational signatures generated by UVR and arsenite differ from those of UVR alone, indicating that arsenite modifies the mutation spectrum rather than simply amplify the UVR signature. Based on our published and preliminary findings, we hypothesize that arsenic enhances UVR-induced skin carcinogenesis by disrupting the zinc finger function of the key DNA repair protein XPA, which in turn, results in deficient nucleotide excision repair leading to greater accumulation of somatic mutations. In Aim 1, we will determine whether exposure to arsenic, UVR or both generates unique mutational signatures and the impact of zinc on identified signatures using whole genome sequencing and advanced computational approaches developed by co-investigator Dr. Alexandrov. Aim 2 will investigate the molecular mechanism of C>T mutation enhancement by arsenic through transcription-coupled nucleotide excision repair inhibition using both biochemical approaches and computational analysis of whole genome sequencing data. In Aim 3, we will use a proven animal model of UVR-induced skin carcinogenesis to define in vivo mutational signatures from UVR- induced tumors with or without arsenic and the impact of zinc on the mutation signature. The outcomes from our rigorously designed studies are expected to provide the first experimental definition of a metal-induced mutation signature and the first analysis of mutational signatures generated by combination exposures to two important and relevant environmental insults, as well as the insights into mechanisms by which arsenic enhances UVR-induced carcinogenesis and how zinc confers protection.